Bone is a frequent site of breast cancer metastasis and relapse, however treatment of bone metastasis remains largely ineffective as the mechanisms underlying its initiation and progression are unclear. Increasing evidence suggests that primary tumors release soluble factors and extracellular vesicles (EVs) that can systemically prime distant metastatic sites to form pre-metastatic niches, which are microenvironments supportive of future tumor cell attachment, survival, and growth. Our preliminary data indicate that breast cancer-derived factors can promote osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs), leading to increased bone matrix production and mineralization. This may be a critical aspect of the bone pre-metastatic niche, as recent studies revealed that breast cancer cells initially localize to regions of active osteogenesis in bone. However, our current understanding of the functional connections between EVs, local osteogenesis, and bone metastasis is limited due in part to a lack of model systems and quantitative approaches that would resolve the spatial and temporal dynamics of pre-metastatic niche establishment in bone. Here, I hypothesize that tumor cell-derived factors promote the development of an osteogenic pre-metastatic niche in bone, which aids in tumor cell colonization of bone. To address this hypothesis, I propose to use a combination of established mouse models, engineered in vitro mineralized models of the bone microenvironment, and innovative imaging and analysis tools for quantitative evaluation of bone formation. In Aim 1, I will characterize the effect of tumor-derived factors on osteogenic activity in bones of mice and in MSC cultures pre-conditioned with tumor factors. I further dissect the respective roles of soluble factors and EVs in mediating the effects of tumor-derived factors on osteogenic activity in vitro. In Aim 2, I will investigate the influence of tumor-derived factor-induced osteogenic activity on tumor cell colonization and proliferation in bone using a mouse model of bone metastasis and the novel application of a bone serial milling technique to analyze the 3D localization of tumor cells with respect to regions of osteogenic activity. Knowledge gained from this work will further our understanding of the specific mechanisms underlying bone metastasis, which may be applicable to other types of bone-metastatic cancers (e.g. prostate, lung, and kidney). Furthermore, this work may directly improve patient outcome by revealing potential therapeutic targets to prevent bone metastasis. !